Toxicity and biochemical impact of methoxyfenozide/spinetoram mixture on susceptible and methoxyfenozide-selected strains of Spodoptera littoralis (Lepidoptera: Noctuidae)

Methoxyfenozide (M) is one of the selective insecticides used in integrated pest management (IPM) programs for lepidopteran pests. However, recent studies reported a development of M-resistance, which prompted us to look for alternatives. Here, we investigate the potency of a mixture of M with spinetoram (Sp) on M-resistant insects. In the laboratory, a selection pressure with M has carried out on Spodoptera littoralis (Lepidoptera: Noctuidae) strains. A dipping technique was used to evaluate the toxicity of a sublethal concentration of M and Sp. on S. littoralis larvae, and the same concentrations were used to assess the toxic impact of their combination on susceptible (SUS) and M-selected (MS) strains. The toxicity of M/Sp mixtures was computed using a combination index equation, and a potentiation effect was observed in the two tested strains. Synergism tests revealed that piperonyl butoxide had considerable synergistic effects on M toxicity in the MS strain. The results revealed that the M/Sp mixture's negative effect on both monooxygenases and esterases is most likely the cause of its potentiation effect on the SUS and MS strains. It was concluded that M/Sp mixtures are effective against M-resistant S. littoralis strains, so these can be used in IPM programs.


Materials and methods
Susceptible strain. The susceptible strain of S. littoralis has established from egg batches collected from a cotton field at the agricultural research and experimental station, Faculty of Agriculture (University of Cairo), in the summer of 2018. Before the experiment began, this colony was reared in the laboratory for twelve generations without being exposed to insecticides. The strain was maintained at 26 °C ± 2 °C and 65% ± 5% relative humidity (RH) with a 16:8 h. light: dark photoperiod 35 . A 10% sugar solution was given to newly emerged moths, and they were allowed to lay their eggs on tissue paper. The collected eggs were maintained for hatching in other jars. Throughout the larval period, fresh castor oil plant leaves, Ricinus communis L., were supplied daily to the larvae. From this culture, second instar larvae were selected for bioassay tests. All experiments were performed in accordance with the relevant guidelines and regulations for use of plants. The castor plant was identified and authenticated by a Botanist at the Botany Department, Faculty of Agriculture, Cairo University, Egypt. Confirmation of the taxonomic identity of the plant was achieved by comparison with voucher specimens kept at the Egyptian Agriculture museum, and the use of documented literature 36 . The official permission of collecting castor plant leaves from greenhouses owned by Cairo university's Faculty of Agriculture for feeding insects and conducting research experiments was obtained from the vice dean for environmental affairs and community services sector.
Selection with methoxyfenozide. The methoxyfenozide-selected strain was derived from a susceptible strain after 16 generations of treatment with 1-70 μg/mL of methoxyfenozide, which was specifically chosen for selection due to the current strain's accelerated rate of developing resistance to methoxyfenozide. Using leaf dipping bioassay technique 37 , the second instar larvae were exposed to the pesticide at a concentration comparable to the LC 50 of the baseline set for the laboratory colony in the first round of selection. Surviving larvae were transferred to untreated castor leaves and reared in the laboratory under the conditions specified above after 24-h exposure. During selection cycles, the mortality ranged from 10 to 90%. Based on the results of the previous generation's bioassays, the methoxyfenozide concentration utilized to select each successive generation was LC 50 . Depending on availability, the number of second instar larvae used for each generation varied (1000-2000).
Bioassay. In three independent experiments, a leaf dipping bioassay technique 37 was used. Lethal concentration (LC) values were determined using a range of five to seven serial concentrations of each insecticide (diluted with tap water). Castor plant leaves were dipped in each prepared concentration for 20 s before drying at room temperature (29 ± 2 °C) for 1 h. One hundred of the second instar larvae of the susceptible strain were placed in glass jars covered with a clean muslin cloth and divided into five replicates (20 larvae/replicate). The larvae were starved for 4 h before feeding and were allowed to feed on the treated leaves for 24 h. Any living larvae were transferred to clean jars with new untreated castor leaves after 24 h. Abbott's formula was used to correct the mortality percentages after 96 h 38 . The toxicity index, which is the ratio between the LC 50 of the most toxic insecticide and the LC 50 of our tested insecticide multiplied by 100, was calculated 39 . For the analysis of synergistic effects, PBO, DEM, and TPP were dissolved in acetone. Toxicity was first determined using a range of synergist concentrations to find a suitable concentration that did not affect larval mortality. Concentrations up to 100 mgL −1 of these synergists had no effect on larval mortality (P > 0.05). After 96 h, larvae mortality was recorded. The synergism ratio (SR) was calculated by dividing the LC 50 of insecticide alone by the LC 50 25 of methoxyfenozide on the methoxyfenozide-selected strain. Each binary mixture was diluted five to seven times in bioassays, with a serial dilution factor of two. Using the same bioassay method described previously, the second instar larvae of susceptible and methoxyfenozideselected S. littoralis strains were subjected to each dilution in three replicate samples. The combination index (CI) 40 was adopted to quantify the potentiation (CI < 1), additive (CI = 1), or antagonistic (CI > 1) effects. Based on the bioassay results, the CI values at 10, 50, and 90 percent mortality rates were calculated using the Com-puSyn software (www. combo syn. com).
where n(CI)X is the combination index for n insecticides at x% mortality rate, (Dx)1 − n is the sum of the concentrations of n insecticides causing x% mortality in insecticide combination, [ in distilled water w/v in 2:5 ratio) was added and the total volume was made up to 1 mL with PPB ( 40 mM, PH 7). The enzyme activity was read at 600 nm as an endpoint (Spectrophotometer UV-VIS, Shimadzu UV-1201), and the absorbance levels were compared with a standard curve of absorbance for known concentrations of α-naphthol (50 mM methanolic stock solution). Three replicates at least for each treatment and control were used. The α-esterase-specific activities were reported as [µmoles of α-naphthol formed min -1 mg -1 protein].
Glutathione-S-transferase assay. After 96 h, twenty larvae of each treatment and control were weighed, rinsed with distilled water, and homogenized in 100 mM potassium phosphate buffer containing 1 mM EDTA at pH 6.5. Then, the homogenates were centrifuged at 10,000 rpm for 10 min. The supernatants were transferred into a clean Eppendorf 42 method was used to determine the Glutathione-S-transferase (GST) activity with some modifications. Briefly, 3 ml of the reaction mixture was made from 150 µL of 50 mM reduced L-glutathione (GSH), 50 µL of 50 mM CDNB, and 30 µL of the sample supernatant. The absorbance increment at 340 nm was recorded at a 1-min interval against a blank for 5 min. An extinction coefficient of 9.6 mM/cm was used to calculate the amount of CDNB conjugated. Three replicates were used to determine the GST activity for each treatment and control. The GST-specific activities were expressed as [nmols min -1 mg -1 protein].
Fluorometric monooxygenase (MO) determination. MO activity was determined using the 43   www.nature.com/scientificreports/ the enzyme assays are presented as mean ± SEM and were analyzed by one-way analysis of variance followed by Tukey's post-hoc test (P < 0.01) using an SPSS software (version 15.0, SPSS Inc., Chicago, IL, U.S.A).

Results
Methoxyfenozide resistance selection. The insects rapidly developed resistance to methoxyfenozide when unceasingly selected with increasing concentrations under laboratory conditions. The LC 50 value was increased to 63.35 mg L −1 after 16 generations of selection, compared to 1.748 mg L −1 for the beginning susceptible colony (Fig. 1). These data indicate that the selected strain developed a 36.2-fold increase in resistance toward methoxyfenozide (M) during the selection processes.
Synergistic effect. Table 1 shows the synergistic effects of PBO, DEM, and TPP with methoxyfenozide against susceptible (SUS) and methoxyfenozide selected (MS) strains of Spodoptera littoralis. The synergists tested did not affect the toxicity of methoxyfenozide in the SUS strain; however, in the MS strain, PBO produced a 3.33-fold synergism. TPP synergy was not observed in either strain. DEM reduced methoxyfenozide toxicity in the SUS strain, but it increased it in the MS strain.
Toxicity of the tested insecticides alone on susceptible and resistant strains.     Table 4. In the laboratory and MS strains, the LC 50 of the M/Sp combination increased from 0.046 to 62.32 µg AI ml −1 , respectively. Despite this, the M/Sp combination demonstrated an extremely strong potentiation in both strains (Table 4).
Detoxification enzymes. Carboxylesterase activity. When compared to the control, the sublethal concentration (LC 25 ) of methoxyfenozide and spinetoram did not affect the alpha-esterase-specific activity (µ moles min -1 mg -1 protein) in either the laboratory (F = 43.2, P < 0.001) or resistant (F = 52.8, P < 0.001) strains, but the M/Sp mixture showed statistically significant inhibition in their activity ( Fig. 2A and B).
Glutathione-S-transferase activity. As shown in Fig. 2C, spinetoram alone and in combination with methoxyfenozide significantly increased the GST-specific activity (nmol min -1 mg -1 protein) in the laboratory strain Table 2. Toxicity (LC values) of methoxyfenozide and spinetoram individually to the 2nd instar larvae of a susceptible of Spodoptera littoralis after 96 h. post-treatment. No. number of larvae exposed to the insecticide, LC 25, and LC 50 are concentrations of each insecticide, in μg (microgram) of insecticide per mL (milliliter) of water, that is required to kill 25 or 50% of the tested population, respectively. X 2 chi-square, and df Degree of freedom. g value goodness of fit, and *Toxicity index 39  www.nature.com/scientificreports/ (F = 63.38, P < 0.001), but methoxyfenozide alone had no effect when compared to the control. The GST-specific activity in the resistant strain did not differ significantly between the treatments (F = 3.25, P > 0.01) (Fig. 2D).
Monooxygenase activity. In the laboratory strain (SUS), the sublethal concentration (LC 25 ) of Sp or methoxyfenozide individually significantly increased the MO activity (pmols min -1 mg -1 protein) (F = 30.8, P < 0.001), but the M/Sp mixture showed statistically significant inhibition in its activity when compared to the control (Fig. 2E). However, in the MS strain, the MO activity did not change statistically when treated with the sublethal concentration of methoxyfenozide compared to the control, but the M/Sp mixture showed statistically significant inhibition in its activity (F = 71.9, P < 0.001) (Fig. 2F).

Discussion
In this study, the methoxyfenozide (M) resistance laboratory-selected S. littoralis showed reduced susceptibility to methoxyfenozide after 16 continuous generations. The LC 50 increased from 1.74 μg/mL in the parent strain to 63.31 μg/mL (Fig. 1). This result agrees with 27 , as they successfully selected a field-collected colony of S. exigua www.nature.com/scientificreports/ for methoxyfenozide resistance after only seven generations. In addition, Moulton et al. 6 reported a 120-fold increase in methoxyfenozide resistance in a field population of S. exigua after a few generations of selection. S. littoralis is a swarming, polyphagous, foliage-feeding insect found worldwide. This insect is one of the most frequent cotton pests, wreaking havoc on various crops 49 . One of the most important problems in this pest is its resistance to almost all chemical groups used against it 50 . Consequently, it has sparked a lot of interest in finding ways to avoid or overcome this problem. For example, insecticide mixtures may present intriguing possibilities for pest management, particularly if potentiation interactions among insecticides occur 51 . As stated by Ahmad 34 , mixing pesticides with different modes of action may delay the development of resistance within pest populations. This is because the resistance mechanisms required for each pesticide in the mixture may not be widely distributed or exist in insect populations 52 .
This study assessed the insecticidal effects of the LC 25  Depending on the LC 50 values of both compounds, they are considered highly toxic to the SUS strain of S. littoralis (Table 2). Furthermore, in the MS strain, there was no cross-resistance between Sp and methoxyfenozide, as the RR for Sp after 16 generations of selection pressure with M was 3 ( Table 3). This finding agrees with 26 , who reported a negative cross-resistance between methoxyfenozide and spinetoram in the MS strain of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae). They suggested using spinetoram to mitigate the methoxyfenozide resistance in the field.
In insects, the detoxification process involves adding functional groups to lipophilic xenobiotics, primarily through oxidation-reduction and/or hydrolysis reactions carried out by phase I enzymes like cytochromes P450s and carboxylesterases (CaEs). Then, phase II enzymes such as GSTs conjugate phase I metabolites into small hydrophilic molecules 53 . These detoxification enzymes, MO, CaEs, and GST, have been reported to gain the most significant role in insect resistance to either synthetic or non-synthetic insecticides 52,54 . Globally, the resistance is mostly associated with increased levels of these detoxifying enzymes in insecticide-resistant populations 55 .
In this study, using the LC 25 values of methoxyfenozide and spinetoram individually did not statistically change the esterase-specific activity compared to the control group in the SUS and MS strains ( Fig. 2A and B), indicating that esterases are insensitive to these compounds. In many insect species, increased esterase activity is a major mechanism of insecticide insensitivity or resistance 56 .
In contrast, the LC 25 values of methoxyfenozide and spinetoram individually elevated the activity of MO enzymes in the SUS and MS strains ( Fig. 2E and F); indicating that these enzymes may have a role in the degradation of these two compounds. methoxyfenozide showed considerable synergism with PBO in the MS strain, which agrees with 28 , indicating that MO was involved in resistance. Metabolic enzymes have been linked to methoxyfenozide resistance in cotton leafworm S. littoralis 5 and H. armigera 57 . Moreover, the involvement of MO in the mechanism of spinosad resistance was reported in S. exigua 58,59 . Sial et al. 60 also recorded the same result when Sp was used against Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae). These results were expected as spinosad and spinetoram are both spinosyns.
One of the intriguing findings in this study is the significant decrease in MO activity after 96 h of treatment with a mixture of sublethal concentrations of methoxyfenozide and spinetoram in both SUS and MS strains ( Fig. 2E and F). This finding suggests that the potency of this mixture may be attributed to the ability of both compounds to disrupt the insect's detoxification metabolic pathway of these compounds. This conclusion is supported by the significant decrease in the activity of esterase enzymes after the treatment with the same mixture in the SUS and MS strains, while the activity of esterase enzymes activity did not change when each compound was used individually.
However, some resistance mechanisms in S. littoralis, such as increased MO detoxification 61 , may nullify the benefits of pesticide combinations. Moreover, mixtures may also give way to new resistances, which may expand to other chemical classes and become challenging to handle 34 . Fortunately, this study found no evidence of M-Sp cross-resistance. This finding, together with the resistant strain's high level of sensitivity to this mixture, implies that using this mixture against S. littoralis is useful in avoiding the rapid development of M resistance.
This study highlighted the importance of testing insecticide mixtures on resistant pest strains. The mixture's success on susceptible strains does not necessarily imply its success on resistant strains, which are typically found in the fields. Additionally, one significant benefit of using the mixture suggested in this study is that both its components are very safe for mammals and non-target organisms, and they do not pollute the environment. It is also expected that using low concentrations of both compounds to manage lepidopteran pests associated with cotton will have no negative effects on biological systems or the environment. However, further research on this mixture is required to test its chronic toxicity to mammals. Furthermore, the GST activity was measured using the conjugation of CDNB, which demonstrated no significant differences between any of the treatments and the unselected colony in the MS strain (Fig. 2D). However, more research should be done using both CDNB and 1,2-dichloro-4-nitrobenzene to see if GSTs are involved in the detoxification process.

Data availability
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